Progress initially was slower than we had hoped because we did not have full tissue culture room facilities until spring, 2011 and then we had to regenerate many of our biological reagents (PKD1-expressing cell lines, some of our cDNA constructs) that we had previously produced at JHU. We also had to systematically re-characterize our various antibodies since the fellow working on this project is new and did not overlap with my JHU team. Despite the delays, we have made considerable progress on a number of fronts: a) We have evidence that there may be a free pool of the N-terminal cleavage product of PC1 (NTF) that may be functional. We are currently working with the NIDDK Proteomics core to identify potential binding partners. b) We have preliminary data linking PC1 to the Arl family of proteins. We are collaborating with Tamara Caspary (Emory) to confirm our results and better define the nature of the relationship. c) The majority of our effort this past year has been focused on studying the earliest stages of cyst formation using our inducible Pkd1 floxed mouse line. We have intensively interrogated the expression status of kidneys as they morph from normal, to pre-cystic to frankly cystic stages and in parallel, correlated these findings with metabolomic studies performed on corresponding urine samples from the same mice (in collaboration with Frank Gonazalez, NCI). We identified HNF4a as a potentially important network node and tested this hypothesis by modifying the HNF4a genotype in Pkd1 mutant mice. We found that deleting HNF4a resulted in significantly worse disease, consistent with the genes role as an important modifier of disease. A manuscript describing these findings has been prepared and is currently being reviewed by our collaborators. We have also identified several other important network nodes and studies are presently underway to verify their role as disease modifiers using genetic and pharmacologic methods. d) We have complemented these efforts with parallel studies focused on the late-onset model of cystic disease. We have better defined the kinetics of late onset disease in this model and while more work is needed our data currently suggest a rapid onset of cyst formation after a long period of normalcy (many months). We have performed extensive gene expression and metabolomic analyses and these results are currently being evaluated. e) We are developing an alternative cell culture system for study of tubule vs cyst formation. We acquired a spontaneously immortalized mouse renal collecting duct cell line from Switzerland whose properties have been well-defined by the investigator. The cells maintain V2R expression and make tubules when cultured in matrix. We have isolated sub-clones that consistently make tubules and used lentiviral delivery systems for siRNA against Pkd2 and Pkd1 to knock-down the respective gene expression. We are presently characterizing the modified lines and in collaboration with the Knepper lab will be comparing V2 receptor signaling in wild type and mutant cells. In parallel, we also will be comparing gene expression patterns and regulatory networks, using pertubation analysis to test the models that are established. f) Other studies in progress: we have crossed the Pkd1 floxed line to a variety of other Cre-lines to examine the role of Pkd1 in other cell types. We also have crossed the tet-inducible Cre system into our Pkd1 floxed line and are presently testing whether we can use this approach to make a better model of human disease with waves of mutation induced at various life stages with fewer problems than are associated with the use of the tamoxifen-inducible Cre recombinases. Finally, we have crossed in several reporter genes for various signaling pathways to look at the effects of Pkd1 inactivation in vivo on cellular signaling. We collaborated with a group in Canada who used this approach to show that Wnt signaling is not upregulated in Pkd1 mutants Miller et al, Kidney International, 2011). g) We have begun characterization of a new Pkhd1 floxed line that we developed in collaboration with Terry Watnick at JHU. We have generated healthy heterozygotes and they are now sufficiently old enough to begin crossing to produce homozygotes. We have targeted the C-terminus of the gene, a region never before manipulated in vivo and which is thought to encode a domain that translocates to the nucleus. In parallel, we have begun crossing this line with the Pkhd1 floxed line that we had previously generated with the goal of getting the loxP sites from each onto the same haplotype so we can induce large-scale gene deletion. This work is in progress. h) We have continued our collaboration with Jun-ya Kaimori (Japan) in characterizing the relationship between polyductin/fibrocystin and Smurfs. We have confirmed many of our previous in vitro findings using primary cells from Pkhd1 mutant rodents and are now investigating the mechanisms responsible for our findings.